SUMMARY HIV-1 is the causative agent of AIDS, which remains a global pandemic. The need continues for more effective and broadly active preventive strategies and anti-retroviral therapies to target infectious virus and already- infected cells. In both virus-cell and cell-to-cell infection, the metastable, virus-specific envelope protein (Env) is activated by cell receptors to enable entry and disease progression. The underlying hypothesis driving Project 4 of this Program Project is that the conserved sites in Env gp120 and the conformational metastability of the Env gp120-gp41 trimer spike can be used as ?Achilles? heels? to target both HIV-1 virus and virus- infected cells. The project will be underpinned initially by a class of peptide triazole (PT) Env antagonists that [1] target a specific gp120 site overlapping the conserved CD4 binding site, [2] suppress gp120 interactions at both of the cell receptor binding sites, [3] drive Env gp120 into an inactivated conformation and [4], upon treatment of HIV-1, cause gp120 shedding and irreversibly inactivate the virus. The work will be stimulated by a major recent breakthrough with the discovery of more drug-like small cyclic PTs (cPTs) that retain the functional signature of the original linear PTs but with massively increased proteolytic resistance. cPT variants with expanded chemical space for future structural variation have also been discovered. A major goal in this project going forward is to advance designs for cPTs through synthesis and mechanistic definition of cPT-Env encounter. We will seek to expand understanding of mechanisms by which cPTs inactivate viruses and Env- expressing cells through Env spike trimer interactions, and seek to integrate this understanding with the evolving definition of Env trimer structures and conformational states. Investigations of cPT inhibitors will examine the notion that PTs and cPTs can hijack the intrinsic conformational metastability properties of the Env protein and the associated metastability of the virus. The successful derivation and properties of cPTs will be used as a springboard to derive new structure-minimized surrogates targeting the conserved region of the gp120 subunits of Env where cPTs bind. In addition, the combined macrocycle-to-peptidomimetic approach will be used to derive antagonists against more diverse Env trimer epitopes for neutralizing the virus. We will relate the mechanisms of peptidomimetic/macrocyclic inhibitors with those for other small molecule inhibitors investigated in this Program. The investigations of Project 4 will address all four broad specific aims of the Program Project team, namely to identify inhibitors of HIV-1 Env, to determine their interaction mechanisms with Env proteins, to determine their effects on virus and cell Env protein trimers and to determine the potential for virus resistance.